Anti-viral compounds

ABSTRACT

Cyclohepta(a)naphthalene derivatives and processes for their preparation. Compounds are mitotic suppressants and active against DNA viruses, for example Herpes simplex.

United States Patent Borrow et al.

[451 Sept. 25, 1973 ANTI-VIRAL COMPOUNDS Assignee: Imperial ChemicalIndustries Limited, London, England Filed: Feb. 28, 1972 Appl. No.:230,140

US. Cl. 260/485 G, 195/51, 260/488 B,

260/617 F, 424/311, 424/313, 424/343 Int. Cl... C07c 35/44, C07c 69/20,C07c 69/40 Field of Search 260/488 B, 485 G,

[56] References Cited OTHER PUBLICATIONS Bull. Soc. Chim. (France) 1959,1128.

Primary ExaminerVivian Garner AttorneyCushman, Darby & Cushman [57]ABSTRACT Cyclohepta[a]naphthalene derivatives and processes for theirpreparation. Compounds are mitotic suppressants and active against DNAviruses, for example Herpes simplex.

2 Claims, N0 Drawings ANTI-VIRAL COMPOUNDS This invention relates to newcompounds which are mitotic suppressants, that-is, they inhibit mitosis,and which are active against DNA viruses, that is, viruses containingdeoxyribonucleic acid, for example Herpes simplex and Vaccinia viruses.

According to the invention there are provided the compound whichpossesses the applicants code number I.C.I. 69653, and the monoacetatethereof, and the hemisuccinate thereof and pharmaceuticallyacceptablesalts thereof.

I.C.I. 69653 has the structure (I) or its mirror image. The compound canbe named systematically as 304,93-dihydroxy-4a,9a-bis(hydroxymethyl)-4fl,l lbB-dimethyl-2,3,4,4aa,5,6,6aB,7,8,9,10,l1,11a,11btetradecahydro-8B,llaB-methano-ll-I-cyclohepta[a]- naphthalene (or the mirror image inwhich a and B are interchanged). The numbering system used is shown inthe carbon skeleton (II). The compound has the molecular formula C H O,.

The monoacetate of I.C.I. 69653, hereinafter referred to by the codenumber I.C.I. 79743, has the molecular formula C H O The hemisuccinateof I.C.I. 69653, hereinafter referred to by the code number I.C.- I.79744, has the molecular formula C I- As suitable salts of I.C.I. 79744there may be mentioned, for example, alkali metal salts, for example thesodium salt.

According to a further feature of the invention there is provided aprocess for the manufacture of LC]. 69653 which comprises cultivating anI.C.I. 69653- producing strain of Cephalosporium aphidicola in asuitable nutrient medium, whereafter the I.C.I. 69653 so produced isisolated from the culture medium.

The process may be carried out under surface culture or sumbergedculture conditions. However, it is to be understood that some of thesaid strains of Cephalosporium aphidicola will only give I.C.I. 69653 onsurface culture, whereas others will give the compound on submergedculture.

A strain which gives I.C.I. 69653 on surface culture is Cephalosporiumaphidicola Petch I.M.I. 68689(ii), which has been deposited at, and isavailable to the public without any restrictions from, The commonwealthMycological Institute, Ferry Lane, Kew, Surrey, England. A descriptionof this strain is as follows:

COLONY APPEARANCE When grown on agar media at room temperature (2022C.), slow growing, not more than 2cm. in 5 days. White, floccose on allmedia tested, with thick, often crinkled, felt on Raper-steep andIsolation medium. Less vigorous growth on Potato/Dextrose, 2 percentMalt, Czapek-Dox or Raulin-Thom agar.

No pigment produced.

VEGETATIVE MYCELIUM Septate, branching, hyaline.

SPORULATION Conidiophores simple 2040,u.m long, arising in whorls of twoor three (occasionally more) from specialised prostrateconidiophore-bearing hyphae often exceeding 300p.m in length. Theconidiophores usually taper to a point from which phialospores arise inheads which can reach 20pm in diameter but which slime down on maturity.

Conidia are oblong-oval, cylindrical or, rarely, somewhat pyriform,always with obtuse ends 4-8 X 1.5-2.5

The media used are mostly those described in the literature, except thatthe isolation medium also used is as described in Journal of GeneralMicrobiology, (1953), 9, (2), 316, but with the Rose bengal omitted.

A strain of Cephalosporium aphidicola which gives I.CI. 69653 onsubmerged culture is Cephalosporium aphidicola Petch I.M.I. 68689(iii),which has likewise been deposited at, and is available to the publicwithout any restrictions from, the said Commonwealth MycologicalInstitute. A description of this strain is as follows:

I COLONY APPEARANCE When grown on agar at room temperature (20-22 C.),slow growing, not more than 2cm. in 5 days. White, floccose on all mediatested with thick, somewhat crinkled, felt on Raper-steep and Isolationagar. Less vigorous growth on Potato/Dextrose, 2 percent Malt,Czapek-Dox and Raulin-Thom agar.

No pigment produced.

VEGETATIVE MYCELIUM Septate, branching, hyaline.

SPORULATION Conidiophores simple 2040p,m long, arising in whorls of twoor three (occasionally more) from specialised, prostrate, conidiophorebearing hyphae, often exceeding 300 111 in length. The conidiophorestaper to a point from which phialospores are budded to produce headswhich can reach 20pm diameter, but which slime down on maturity.

Conidia are oblong-oval, cylindrical, rarely pyriform, 3-7 X 1-2.5 ,u.m.

The surface or submerged cultivation process of the invention is carriedout in conventional manner. The nutrient medium contains assimilablesources of carbon, nitrogen, phosphorus, magnesium, sulphur andpotassium. Also, the nutrient medium preferably contains minutequantities of the so-called trace elements, i.e. iron, manganese, zinc,molybdenum and copper. The carbon source may be, for example, a sugar,for example glucose, and this source may be present in the medium in aconcentration in the range 01-30 percent by weight, and preferably 5-15percent by weight. The nitrogen source may be an inorganic source or anorganic source, for example sodium nitrate, ammonium tartrate or cornsteep liquor. The nitrogen source may be present in the medium insufficient amount to provide 0.01 to 0.5 percent by weight of elementarynitrogen, and preferably 0.05 to 03 percent by weight of elementarynitrogen. The sources of phosphorus, magnesium, sulphur and postassiummay be, for example, potassium dihydrogen phosphate, magnesium sulphate,a soluble sulphate such as magnesium sulphate, and potassium chloride,respectively. The process may be carried out at l838 C., and preferably-27 C.

At the completion of the cultivation process, the I.C.I. 69653 presentin the culture medium may be isolated by filtration of the culturemedium, adjustment of the culture filtrate to a pH of about 6.5,extraction of the culture filtrate with a suitable organic solvent, forexample chloroform, drying of the organic solution, removal of thesolvent by evaporation, and, if desired, crystallisation of the LC].69653 so obtained from a suitable solvent, for example ethyl acetate or50 percent v/v aqueous acetic acid. I

According to a further feature of the invention there is provided aprocess for the manufacture of I.C.I. 79743, which comprisesmono-acetylating I.C.I. 69653.

As a suitable acetylating agent there may be mentioned, for example,acetic anhydride. The reaction may be carried out in a suitable organicsolvent, for example dry pyridine.

According to a further feature of the invention there is provided aprocess for the manufacture of l.C.l. 79744 andpharmaceutically-acceptable salts thereof, which comprises reactingI.C.l. 69653 with an acylating agent derived from succinic acid, forexample succinic anhydride.

The reaction may be carried out in a suitable organic solvent, forexample dry pyridine.

The biological activities of the compounds of the invention have beendemonstrated in standard biological test procedures. In particular,their mitotic suppressant activity has been demonstrated by exposingcultured mammalion cells (Earles L strain of mouse fibroblasts) tovarying concentrations of the compound for 24 hours. The cells aresubsequently fixed and stained, and are examined under the microscope todetermine the effect of the compound on mitotic rate. The anti- (DNA)viral activity has been demonstrated in vitro and in vivo (in the rabbiteye).

When a compound of the invention is to be administered parenterally to ahost, for example a human patient, in need of mitotic suppressant actionor in need of action against a condition or disease caused, in part atleast, by a DNA virus, the total daily dose may be l0100mg./kg. of thecompound. Alternatively, one of the said compounds, for example in aconcentration of l10mg./ml., may be applied topically as necessary tothe said host.

According to a further feature of the invention there are providedpharmaceutical compositions comprising I.C.l. 69653, I.C.I. 79743, or[Cl 79744 or a pharmaceutically-acceptable salt thereof, and an inert,non-toxic, pharmaceutically-acceptable diluent or carrier.

The pharmaceutical compositions of the invention may be in a formsuitable for parenteral adminsitration, for example sterile injectablesolutions or suspensions, or in a form suitable for administration aseye-drops, for example a ballmilled suspension in the presence of adispersing agent, a suspension with l% hydroxypropylmethylcelluloseadded, or a solution in dimethylsulphoxide. The pharmaceuticalcompositions of the invention are obtainable in a conventional mannerusing conventional diluents and carriers, and they may contain between10 percent by weight and percent by weight of the active ingredient. Thepharmaceutical compositions of the invention may contain between lOmg.and 250mg. of the active ingredient. Suspensions and solutions maycontain between 0.lmg. and 50mg. per ml. of the active ingredient.

The pharmaceutical compositions of'the invention may contain, inaddition to the abovementioned active ingredient, at least one agentwhich is known to be active against DNA viruses, for example idoxuridine(5-iodo-2-deoxyuridine).

The invention is illustrated by the following Examples:

EXAMPLE 1 A nutrient medium was made up as follows:

Sodium nitrate 2.0g. Potassium dihydrogen phosphate 1.0g. Magnesiumsulphate heptahydrate 0.5g. Potassium chloride 0.5g. Ferrous sulphateheptahydrate 0.0lg. Glucose ("Cerelose"', "Cerelose" is a registeredtrade mark) 50.0g. Yeast extract (Oxoid" brand; Oxoid" is a registeredtrade mark) 1.0g. Minor element concentrate (see below) l.0ml.

Distilled water to l l.

The minor element concentrate was made up as follows:

The compounds:

Ferrous sulphate heptahydrate 1.0g. Copper sulphate pentahydrate 0.l5g.Zinc sulphate 1.0g. Manganese sulphate tetrahydrate 0.1g. Potassiummolybdate 0.1g.

were dissolved in distilled water, sufficient concentrated hydrochloricacid was added to give a clear solution, and this was diluted to l l.with distilled water.

The nutrient medium was adjusted to pH 5.8 with ION aqueous potassiumhydroxide and then autoclaved at 15 p.s.i. for 25 minutes.

Cephalosporium aphidicola Petch I.M.l. 68689(ii) was grown at atemperature of 25 C. in surface culture in 200 bottles (capacityone-third pint, i.e., ml.) each containing 30ml. of the nutrient medium.The product was harvested after 31 days. The culture filtrate (2.73 l.)was acidified with aqueous ZN-hydrochloric acid to pH 6.5 and extractedfour times with chloroform (560ml. each time). The combined chloroformextracts were dried with anhydrous sodium sulphate, and then evaporatedto about 100ml. at 50 C'./60mm. The concentrate was kept at 2 C. for 16hours, and the colourless crystals which formed were collected byfiltration. The solid residue was crystallised from 50 percent v/vaqueous acetic acid, and the resulting crystals dried at 40C./2mm.Recrystallisation of these crystals from ethyl acetate gave l.C.I.69653. The compound had the following characteristics:

a m.p. 227-232 C.

b [01],, 11.84 (c, 1g./l00ml. in methanol) c molecular formula C ,,H O

d Analysis: Found C, 70.6, H, 10.0, N, nil. Calc. for c,,,ri,,o,, c,70.97, H, 10.13, N, nil.

e Mass spectrum: Peak at m/e 307 c n o and m/e 320 (G i-1 Correspondingto 0 11 0 minus -CH OH andH O, respectively).

f Infra-red spectrum: max 3490, 3410, 3330, 1077, 1049, 1027 and 966cm(in Nujol) g ULtra-violet spectrum: No absorption in the region 200400nm(2.13mg. in 100ml. of methanol) 7 h Thin layer chromatography. R 0.35 onsilica gel GF (solvent system: 2 percent v/v glacial acetic acid, 3percent v/v acetone, 5 percent v/v methanol, and 90 percent v/v ethylacetate; indicator: chromic acid, followed by heat the spot is firstpink and becomes black).

i Upon treatment with acetic anhydride in pyridine at 22 C. for 50minutes, l.C.l. 69653 forms a diacetate of m.p.163.5-165.5 C. Calc. forC I-1 0 C, 68.2, H, 9.1. Found C, 67.9, H, 8.9.

j Upon treatment with acetic anhydride in pyridine for 16 hours at.22C., l.C.l. 69653 forms a triacetate of m.p.145147 C. Calc. for C ,,H O,,C, 67.2, H, 8.7. Found C, 67.2, H, 8.8.

k Upon treatment with periodic acid in aqueous acetic acid as describedbelow, l.C.l. 69653 gave a compound of m.p.l52156 C.

.To a stirred mixture of 1.C.1. 69653 (200mg) in glacial aceticacid'(12.4m1.) and water (6.2ml.), maintained at 22 C., there was added0.87ml. of a 50 percent w/w solution of periodic acid (H 1O .H,O) inwater. After 10 minutes the mixture was diluted with water (l20ml.) andextracted with chloroform (4 X 120m1.). The extracts were dried overanhydrous sodium sulphate, and then concentrated under reduced pressureto an oil which was separated by chromatography on silica gel (25g) 9:1chloroform-petroleum ether (b.p.60-80 C.) eluted a solid which afterthree crystallisations from a mixture of petroleum ether (b.p.60-80 C.)and ethyl acetate had m.p.l52-l56 C.

1 Upon treatment with chromium trioxide in acetic acid as describedbelow, l.C.l. 69653 gave a compound of m.p.172-175 C.

To a stirred solution of l.C.l. 69653 (500mg) in glacial acetic acid(20ml.) at 22C. were added dropwise, over a period of 40 minutes, ml. ofa solution containing chromium trioxide (2g.) and concentrated sulphuricacid (3.02g.) in water (ml.). After the reaction mixture had beenstirred for a further hour at 22 C., ethanol (10ml.) was added and themixture was extracted with ether (4 X l00ml.). The ethereal extractswere dried over anhydrous sodium sulphate, and then concentrated to givean oil which solidified. Two crystallisations of the solid from amixture of ethyl acetate and petroleum ether (b.p.6080 C.) gavecolourless needles of m.p.172175 C.

Example 2 Cephalosporium aphidicola Petch l.M.l. 68689(iii) was used toinoculate agar slants containing the following medium:

Sucrose 3g./l. Dextrin i5g./l. Urea 0.1g./l. Sodium chloride 0.5g./l.Postassium dihydrogen phosphate 0.5g./l. Yeast extract 1.0g./l. Peptone5.0g./l. Ferrous sulphate heptahydrate 0.01 g./l. Agar 20.0g./l.

The medium was sterilised by autoclaving at 15 p.s.i. for 20 minuteswithout adjustment of pH. The final pH was 6.5-6.6. The slants wereincubated at 25 C. for 4-7 days.

Aliquots of the organism from the agar slants were used to inoculate ml.of nutrient medium in 500ml. conical flasks. The medium in the flaskshad the same composition as that described in Example 1. The flasks weresterilised by autoclaving at 15 p.s.i. for 20 minutes. The flasks wereincubated at 25 C. for 3 days on a rotary shaker with 2 inches throw,running at 240 r.p.m.

The contents of one flask were used to inoculate a stirred fermentercontaining 30 1. of the following medium:

Sucrose 40g./l. Sodium nitreate 2.2g./l. Potassium dihydrogen phosphate5 g./l. Magnesium sulphate heptahydrate 1g./l. Potassium chloride0.5g./l. Minor element concentrate 0.2%

The medium was sterilised at 15 p.s.i. for 30 minutes. The fermenter wasa fully baffled tank, the agitator running at 320 r.p.m., with sterileair supplied at 15 l./min. The culture was incubated at 25 C. for 17days.

The mycelium was removed by filtration, yielding 22 1. of filtrate at pH4.1. The pH was adjusted to pH 6.5 with SN-sodium hydroxide, andextracted twice with 5.5 l. of chloroform. The chloroform extract wasdried over sodium sulphate. The filtrate was concentrated atapproximately 45 C. in vacuo to approximately 100ml., and left overnightat room temperature. The resulting mixture was filtered, and there wasthus obtained l.C.l. 69653.

EXAMPLE 3 To a stirred solution of l.C.l. 69653 (2g) in dry pyridine(25ml.) was added acetic anhydride (0.302g.) in dry pyridine (5ml.). Themixture was stirred at room temperature for 17 hours before addition ofa further portion of acetic anhydride (0.302g.) in dry pyridine (5ml.).The mixture was stood at room temperature for 3 hours. Water (l0ml.) wasthen added, and the mixture was stirred for a further 3 hours. At theend of this period water (5ml.) and a solution of 50 percent w/w aqueousperiodic acid (17.3ml.) were added. The mixture was stirred for 15minutes, acidified to pH 2 with 30 percent sulphuric acid and extractedwith ethyl acetate (5 X 20ml.). The organic extracts were combined,washed successively with 3N sodium hydroxide (4 X 15ml.) and water (2 X25ml.). and then dried over sodium sulphate. Evaporation of the ethylacetate yielded a solid which, after two recrystallisations from ethylacetate, gave 9a-acetoxymethyl-3a,9[3-dihydroxy-4a- 7 hydroxymethyl-4fi,2,3,4,4aa,5,6,6aB,7,8,9,10,11,11a,l1btetradecahydro-8B,1laB-methano-lH-cyclohepta[a]-naphthalene (or the mirror image in which a and B are interchanged),code number 1.C.I. 79743, as colourless needles of m.p.193.5196 C.(Found: C, 69.3,1-1, 9.3 percent, C l-1 requires C, 69.4, H, 9.5percent).

EXAMPLE 4 Succinic anhydride (1.48g.) was added portionwise over aperiod of 24 hours to a stirred solution of 1.C.1. 69653 (g) in drypyridine (75ml.) at room temperature. After the mixture had been stirredfor an additional 22 hours, water (50ml.) and 50 percent w/w aqucousperiodic acid (42.5ml.) were added. The solution was stirred forminutes, acidified to pH 2 with 30 percent sulphuric acid, and extractedwith ethyl acetate (l X 200 and 2 X 150ml.) The combined extracts werewashed with water (l00ml.) and then extracted with saturated sodiumcarbonate solution (2 X 100ml.). The combined alkaline extracts werewashed with ethyl acetate (100ml.), and the organic washing wasdiscarded. The alkaline layer was acidified to pH 2 with 30 percentsulphuric acid and then extracted with ethyl acetate (1 X 200, 2 X150ml.). The combined organic extracts were washed with water (2 X200ml.), dried over sodium suphate, then concentrated at 50 C. in vacuoto a pale yellow oil (4.6g.) which was redissolved in a small volume ofethyl acetate and set aside to crystallise. There was obtained in thisway 9a-(3-carboxypropionyloxymethyl)-3a,9B-dihydroxy-4ahydroxymethyl-4B,llbB-dimethyl- 2,3,4,4aa,5,6,6afi,7,8,9,10,11,11a,l1btetradecahydro-8B,11aB-methano-1H-cyclohepta[a]- naphthalene (or the mirror image in whicha and B are interchanged), code number 1.C.1. 79744, as colourlesscrystals of m.p.142-146 C.

A stirred solution of I.C.l. 79744 (0.2g.) in 50 percent aqueousmethanol (50ml.) was titrated with 0.1 N sodium hydroxide (4.55ml.)until the pH of the solution was 8.0. Most of the methanol was removedin llbB-dimethylvacuo at 30 C. and the resulting solution was filteredand then freeze-dried. There was obtained in this way the sodium salt of1.C.1. 79744 as a colourless solid.

EXAMPLE 5 A dispersing agent of the following composition was prepared:

N onylphenol ethylene oxide condensate (Lissapol' NX: Lissapol' is atrade mark) lml. Sodium salt of sulphated cetyl/oleyl alcohol mixture(Lissapol C) lg. Polyglyceryl n'cinoleate, 30pereent w/v in water(Dispersol O.G.; Dispersolis a trade mark) 3.3ml. Water to 1 litre the9a-monacetate thereof, the 9a-hemisuccinate thereof and pharmaceuticallyacceptable salts of said hemisuccinate.

2. A hemisuccinate salt as claimed in claim 1 which is an alkali metalsalt.

2. A hemisuccinate salt as claimed in claim 1 which is an alkali metalsalt.